Direct in Vivo Intracellular Selection of Conformation-sensitive Antibody Domains Targeting Alzheimer's Amyloid-β Oligomers

The development of conformation-sensitive antibody domains targeting the misfolding beta amyloid (Aβ) peptide is of great interest for research into Alzheimer's disease (AD).We describe the direct selection, by the Intracellular Antibody Capture Technology (IACT), of a panel of anti-Aβ single chain Fv antibody fragments (scFvs), targeting pathologically relevant conformations of Aβ. A LexA-Aβ1-42 fusion protein was expressed in yeast cells, as the “intracellular antigen”. Two different scFv antibody libraries (Single Pot Libraries of Intracellular Antibodies, SPLINT) were used for the intracellular selections: (i) a naïve library, derived from a natural, non-immune, source of mouse antibody variable region (V) genes; and (ii) an immune library constructed from the repertoire of antibody V genes of Aβ-immunized mice. This led to the isolation of 18 different anti-Aβ scFvs, which bind Aβ both in the yeast cell, as well as in vitro, if used as purified recombinant proteins. Surprisingly, all the anti-Aβ scFvs isolated are conformation-sensitive, showing a high degree of specificity towards Aβ oligomers with respect to monomeric Aβ, while also displaying some degree of sequence-specificity, recognizing either the N-terminal or the C-terminal part of Aβ1-42; in particular, the scFvs selected from Aβ-immune SPLINT library show a relevant N-terminal epitope bias. Representative candidates from this panel of the anti-Aβ scFvs were shown to recognize in vivo-produced Aβ “deposits” in histological sections from human AD brains and to display good neutralization properties, significantly inhibiting Aβ oligomer-induced toxicity and synaptic binding of Aβ oligomers in neuronal cultured cells. The properties of these anti-Aβ antibody domains, as well as their direct availability for intra- or extra-cellular “genetic delivery” make them ideally suited for new experimental approaches to study and image the intracellular processing and trafficking of Aβ oligomers.     

Development and Implementation of a Single-Chain Fv Antibody for Specific Detection of Bacillus anthracis Spores

A single-chain Fv (scFv) antibody was developed and applied for efficient and specific detection of Bacillus anthracis spores. The antibody was isolated from a phage display library prepared from spleens of mice immunized with a water-soluble extract of the outer membrane of the B. anthracis spore (exosporium). The library (7 × 10⁶ PFU) was biopanned against live, native B. anthracis ATCC Δ14185 spores suspended in solution, resulting in the isolation of a unique soluble scFv antibody. The antibody was affinity purified and its affinity constant (3 × 10⁸ ± 1 × 10⁸ M⁻¹) determined via flow cytometry (FCM). Preliminary characterization of scFv specificity indicated that the scFv antibody does not cross-react with representatives of some phylogenetically related Bacillus spores. The potential use of scFv antibodies in detection platforms was demonstrated by the successful application of the soluble purified scFv antibody in enzyme-linked immunosorbent assays, immunofluorescence assays, and FCM.     

Development of Lateral Flow Immunoassay for Antigen Detection in Human Angiostrongylus cantonensis Infection

Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis.     

Synthetic Fab fragments that bind the HIV-1 gp41 heptad repeat regions

Recent work has demonstrated that antibody phage display libraries containing restricted diversity in the complementarity determining regions (CDRs) can be used to target a wide variety of antigens with high affinity and specificity. In the most extreme case, antibodies whose combining sites are comprised of only two residues – tyrosine and serine – have been identified against several protein antigens. [F.A. Fellouse, B. Li, D.M. Compaan, A.A. Peden, S.G. Hymowitz, S.S. Sidhu, J. Mol. Biol. 348 (2005) 1153–1162.] Here, we report the isolation and characterization of antigen-binding fragments (Fabs) from such “minimalist” diversity synthetic antibody libraries that bind the heptad repeat regions of human immunodeficiency virus type 1 (HIV-1) gp41. We show that these Fabs are highly specific for the HIV-1 epitope and comparable in affinity to a single chain variable fragment (scFv) derived from a natural antibody repertoire that targets the same region. Since the heptad repeat regions of HIV-1 gp41 are required for viral entry, these Fabs have potential for use in therapeutic, research, or diagnostic applications.     

Cloning and characterization of novel tumor-targeting immunocytokines based on murine IL7

We generated and characterized novel antibody-cytokine fusion proteins (“immunocytokines”) based on murine interleukin-7 (IL7), an immunomodulatory protein which has previously shown anti-cancer activity in preclinical models and whose human counterpart is currently being investigated in clinical trials. The sequential fusion of the clinical-stage antibody fragment scFv(F8), specific to a tumor-associated splice isoform of fibronectin, yielded an immunocytokine (termed “F8-mIL7”) of insufficient pharmaceutical quality and in vivo tumor targeting performance, with a striking dose dependence on tumor targeting selectivity. By contrast, a novel immunocytokine design (termed “F8-mIL7-F8”), in which two scFv moieties were fused at the N- and C-terminus of murine IL7, yielded a protein of excellent pharmaceutical quality and with improved tumor-targeting performance [tumor: blood ratio = 16:1, 24 h after injection]. Both F8-mIL7 and F8-mIL7-F8 could induce tumor growth retardation in immunocompetent mice, but were not able to eradicate F9 tumors. The combination of F8-mIL7-F8 with paclitaxel led to improved therapeutic results, which were significantly better compared to those obtained with saline treatment. The study indicates how the engineering of novel immunocytokine formats may help generate fusion proteins of acceptable pharmaceutical quality, for those immunomodulatory proteins which do not lend themselves to a direct fusion with antibody fragments.     

A slowing effect on visual search by advance information in pigeons (Columba livia): a comparison with humans (Homo sapiens)

A comparative study was conducted to investigate whether the search for a target letter was facilitated when the target and prime (preceding stimulus) letters were identical. Pigeons (Section 2) and human participants (Section 3) were first trained to search for “A” among “Y”s and “E” among “D”s in a condition in which a square shape appeared as the prime (Neutral condition). In subsequent testing, a prime was identical either to the corresponding target (Target-priming condition) or to the distractor (Distractor-priming condition). Humans and pigeons responded differently to the two priming conditions. On early trials, the Target prime facilitated search in humans, reducing reaction times (RTs) to targets. In pigeons, however, RTs were longer with Target primes, suggesting that pre-exposure to target letters may directly inhibit the search for targets in subsequent search displays. Furthermore, pre-exposure to the distractor letters may inhibit the processing of the distractor. On later trials, RTs of humans were faster in both priming conditions than in the Neutral condition, suggesting that expectation of a target facilitated search (“Y” predicted “A” and “D” predicted “E”). In contrast, the pigeons showed no evidence of expectation-based facilitation, with constant slowing effects of the Target prime extending across sessions. Possible mechanisms underlying such a slowing priming effect in pigeons were discussed.     

Light-chain shuffling from an antigen-biased phage pool allows 185-fold improvement of an anti-halofuginone single-chain variable fragment

Halofuginone is an antiprotozoal drug used in the treatment of coccidiosis in poultry, a contagious enteric disease caused by parasites of the Eimeria spp. To ensure that food is free from any halofuginone residues and safe for human consumption, a rapid method to detect these residues below the maximum residue limits (MRLs) in a variety of matrices is necessary. To address this need, we constructed an immune single-chain variable fragment (scFv) library from the RNA of a halofuginone-immunized chicken and selected halofuginone-specific scFv by phage display. The best clone isolated from the library had a limit of detection of 30 ng/ml as determined by enzyme-linked immunosorbent assay (ELISA). However, the minimum MRL for halofuginone in certain foodstuffs can be as low as 1 ng/ml, well below the sensitivity of the selected antibody. The selected antibody was then affinity maturated by light-chain shuffling to further improve the antibody’s assay performance. The halofuginone-specific heavy-chain pool of the biopanned library was assembled with the light-chain repertoire amplified from the original prepanned library. This resulted in a heavy-chain-biased library from which an scFv with the potential to detect halofuginone residues as low as 80 pg/ml was isolated, a 185-fold improvement over the original scFv. This new chain-shuffled scFv was incorporated into a validated ELISA (according to Commission Regulation 2002/657/EC) for the sensitive detection of halofuginone in spiked processed egg samples.     

Generation of High-Affinity Chicken Single-Chain Fv Antibody Fragments for Measurement of the Pseudonitzschia pungens Toxin Domoic Acid

Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single V_H and V_L genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.     

Generation of high-affinity chicken single-chain Fv antibody fragments for measurement of the Pseudonitzschia pungens toxin domoic acid

Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single V(H) and V(L) genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.     

Construction and characterization of a pseudo-immune human antibody library using yeast surface display

Lymphocytes from eight individuals out of 60 healthy donors, whose plasmas showed relatively higher antibody titer for a target antigen of death receptor 5 (DR5), were selected for the source of antibody genes to construct so called an anti-DR5 pseudo-immune human single-chain fragment variable (scFv) library on the yeast cell surface (approximately 2x10(6) diversity). Compared with a large nonimmune human scFv library (approximately 1x10(9) diversity), the repertoire of the pseudo-immune scFv library was significantly biased toward the target antigen, which facilitated rapid enrichments of the target-specific high affinity scFvs during selections by fluorescence activated cell sortings. Isolated scFvs, HW5 and HW6, from the pseudo-immune library showed much higher specificity and affinity for the targeted antigen than those from the nonimmune library. Our results suggest that a pseudo-immune antibody library is very efficient to isolate target-specific high affinity antibody from a relatively small sized library.     

Single cell-level detection and quantitation of leaky protein expression from any strongly regulated bacterial system

Extremely low levels of “leaky” expression of genes in bacterial protein expression systems can severely curtail cell viability when expressed proteins are toxic. A general method for sensitive detection of such expression is lacking. Here, we present a method based on microscopic visualization of a fluorescent “reporter” protein (RFP–HU-A) constructed by fusing red fluorescent protein (RFP) to the N-terminus of a nucleoid-associated, histone-like DNA-binding protein, HU-A. Localization of RFP–HU-A within nucleoids facilitates detection, quantitation, and characterization of leaky expression at the single-cell level.     

Production of a soluble single-chain variable fragment antibody against okadaic acid and exploration of its specific binding

Okadaic acid is a lipophilic marine algal toxin commonly responsible for diarrhetic shellfish poisoning (DSP). Outbreaks of DSP have been increasing and are of worldwide public health concern; therefore, there is a growing demand for more rapid, reliable, and economical analytical methods for the detection of this toxin. In this study, anti-okadaic acid single-chain variable fragment (scFv) genes were prepared by cloning heavy and light chain genes from hybridoma cells, followed by fusion of the chains via a linker peptide. An scFv–pLIP6/GN recombinant plasmid was constructed and transformed into Escherichia coli for expression, and the target scFv was identified with IC–CLEIA (chemiluminescent enzyme immunoassay). The IC₁₅ was 0.012 ± 0.02 μg/L, and the IC₅₀ was 0.25 ± 0.03 μg/L. The three-dimensional structure of the scFv was simulated with computer modeling, and okadaic acid was docked to the scFv model to obtain a putative structure of the binding complex. Two predicted critical amino acids, Ser32 and Thr187, were then mutated to verify this theoretical model. Both mutants exhibited significant loss of binding activity. These results help us to understand this specific scFv–antigen binding mechanism and provide guidance for affinity maturation of the antibody in vitro. The high-affinity scFv developed here also has potential for okadaic acid toxin detection.     

A peptide-based fluorescent ratiometric sensor for quantitative detection of proteins

A ratiometric fluorescent sensor was obtained by solid-phase synthesis of a peptide singly labeled at its N-terminus with a 3-hydroxychromone (3HC) derivative, an environmentally sensitive fluorophore with a two-band emission. The construct contains the binding site recognized by an antibody fragment, scFv1F4_Q34S, with nanomolar (nM) affinity. The dye only marginally affected the kinetic and equilibrium binding parameters of the scFv-peptide interaction, as measured by surface plasmon resonance. On interaction with the antibody fragment, the sensor showed up to 47% change in the ratio of its two emission bands, indicating an enhanced screening of the 3HC fluorophore from bulk water. Competition with two unlabeled peptides of different lengths led to a dynamic displacement of the construct governed by the relative binding constants. Calibration showed that the response is proportional to the ratio of scFv1F4_Q34S to labeled peptide. The detection limit of scFv1F4_Q34S was 15 nM. In a more complex medium (100 μg/ml bovine serum albumin), the scFv could be detected in the 50- to 100-nM range. This work demonstrates that, with the perspective of further improvements of the dye spectroscopic properties, fluorescent ratiometric sensing based on small synthetic peptides represents a promising tool for quantitative target detection.     

The distribution of the coalescence time and the number of pairwise nucleotide differences in a model of population divergence or speciation with an initial period of gene flow

This paper is concerned with a model of “isolation with an initial period of migration”, where a panmictic ancestral population split into n descendant populations which exchanged migrants symmetrically at a constant rate for a period of time and subsequently became completely isolated. In the limit as the population split occurred an infinitely long time ago, the model becomes an “isolation after migration” model, describing completely isolated descendant populations which arose from a subdivided ancestral population. The probability density function of the coalescence time of a pair of genes and the probability distribution of the number of pairwise nucleotide differences are derived for both models. Whilst these are theoretical results of interest in their own right, they also give an exact analytical expression for the likelihood, for data consisting of the numbers of nucleotide differences between pairs of DNA sequences where each pair is at a different, independent locus. The behaviour of the distribution of the number of pairwise nucleotide differences under these models is illustrated and compared to the corresponding distributions under the “isolation with migration” and “complete isolation” models. It is shown that the distribution of the number of nucleotide differences between a pair of DNA sequences from different descendant populations in the model of “isolation with an initial period of migration” can be quite different from that under the “isolation with migration model”, even if the average migration rate over time (and hence the total number of migrants) is the same in both scenarios. It is also illustrated how the results can be extended to other demographic scenarios that can be described by a combination of isolated panmictic populations and “symmetric island” models.     

Tail variation of the folding primer affects the SmartAmp2 process differently

Folding primer (FP), together with turn-back primer (TP) and boost primer (BP), is one of the major components of SmartAmp2, a rapid amplification-based method for SNP detection. FP has a unique design where the annealing region is combined with a tail that can fold back. FP tails can be classified as either “strong” or “weak”, depending on the melting temperature and free energy of the hairpin structure. We report that FP tails affect the amplification process differently; by changing the FP concentration, we can increase the amplification reaction speed with “strong tails”. Unlike “strong tails”, concentration change of FP with “weak tails” did not show significant impact on the amplification speed. The comparative analyses using gel electrophoresis demonstrate that the FP type and FP ratio in the reaction change the amplification pattern. The above observations can be used to optimize the reaction and manipulate the reaction speed of SmartAmp2.     

Peach fruit ripening: A proteomic comparative analysis of the mesocarp of two cultivars with different flesh firmness at two ripening stages

A proteomic analysis was conducted on peach fruit mesocarp in order to better elucidate the biochemical and physiological events which characterize the transition of fruit from the “unripe” to the “ripe” phase.The first goal of the present work was to set-up a protocol suitable for improving protein extraction from peach mesocarp. The use of freeze-dried powdered tissue, together with the addition of phenol prior to the extraction with an aqueous buffer, significantly increased the protein yield and the quality of 2-DE gels. The proteomic profiles of the mesocarp from peach fruit of a non-melting flesh (NMF; ‘Oro A’) and a melting flesh (MF; ‘Bolero’) cultivar, at “unripe” and “ripe” stages as defined by some parameters typical of ripening, were then analyzed.The comparative analysis of the 2-DE gels showed that in NMF and MF peaches the relative volumes of 53 protein spots significantly changed in relation to both the ripening stage (“unripe” versus “ripe”) and/or the genetic background of the cultivar (‘Oro A’ versus ‘Bolero’).Thirty out of the 53 differently abundant spots were identified by LC-ESI-MS/MS. The analysis revealed enzymes involved in primary metabolism (e.g. C-compounds, carbohydrates, organic acids and amino acids) and in ethylene biosynthesis as well as proteins involved in secondary metabolism and responses to stress.Among these, 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) appeared to be one of the proteins with the largest change in relative abundance during the fruit transition from the pre-climacteric (“unripe”) to the climacteric (“ripe”) phase. Other proteins, such as S-adenosylmethionine synthetase and β-cyanoalanine synthase involved in ethylene metabolism, were also identified. Moreover, the changes in the relative abundances of a sucrose synthase and an α-amylase suggested differences between the two cultivars in the carbohydrate import activity of ripe fruit. The different accumulation of a few typical ROS-scavenger enzymes suggested that a higher oxidative stress occurred in MF with respect to NMF fruit. This result, together with data concerning the levels of total proteins and free amino acids and those regarding proteins involved in the maintenance of tissue integrity, was consistent with the hypothesis that the last phase of ripening in MF fruit is characterized by the appearance of a senescence status.The present study appears to define well some of the biochemical and physiological events that characterize the ripening of peach and, at the same time, provides interesting indications that could be employed in future marker assisted selection (MAS) programmes aimed to obtain MF fruits with higher ability to preserve tissue functionality maintaining for a longer time their organoleptic characteristics.     

Configural processing of different topologically structured figures: an ERP Study

According to Chen’s theory, topological differences are perceived faster than feature differences in early visual perception. We hypothesized that topological perception is caused by the sensitivity in discriminating figures with and without “holes”. An ERP experiment was conducted utilizing a passive paradigm to investigate the differences in perceiving figures with “hole” and with “no-hole”. The results showed differences in N170 components between figures with “holes” and with “no-holes”. The inversion of the “hole” could influence the latency of N170, but the inversion of the “no-hole” could not, which indicated that global features are processed first in the “hole” perception whilst local features are given priority to the “no-hole” perception. This result was similar to studies concerning face and non-face objects, suggesting a configural processing of the “hole”.     

Optimal control for pandemic influenza: The role of limited antiviral treatment and isolation

The implementation of optimal control strategies involving antiviral treatment and/or isolation measures can reduce significantly the number of clinical cases of influenza. Pandemic-level control measures must be carefully assessed specially in resource-limited situations. A model for the transmission dynamics of influenza is used to evaluate the impact of isolation and/or antiviral drug delivery measures during an influenza pandemic. Five pre-selected control strategies involving antiviral treatment and isolation are tested under the “unlimited” resource assumption followed by an exploration of the impact of these “optimal” policies when resources are limited in the context of a 1918-type influenza pandemic scenario. The implementation of antiviral treatment at the start of a pandemic tends to reduce the magnitude of epidemic peaks, spreading the maximal impact of an outbreak over an extended window in time. Hence, the controls’ timing and intensity can reduce the pressures placed on the health care infrastructure by a pandemic reducing the stress put on the system during epidemic peaks. The role of isolation strategies is highlighted in this study particularly when access to antiviral resources is limited.     

A preliminary chemotaxonomic study on the condensed tannins of green banana flesh in the Musa genus

Sixty percent acetone-soluble tannins were obtained from the flesh of 29 accessions of green bananas and plantains belonging to the AA, AB, AAA, AAB, and ABB genotypes. Their tannin contents were between 0.3 and 2.1% of fresh matter, except for the Pacific plantains (“Maoli” and “Pōpō’ulu” sub-groups) which were tannin-free. (−)-Epigallocatechin (EGC) was their major constituent (88–98%) followed by minor proportions of (−)-epicatechin (EC) (2–12%). Hierarchical average linkage clustering analysis using (EGC/EC) as the sole variable agglomerated all plantains (AAB), while the “Pisang Lilin” cultivar (AA, “Pisang Lilin” sub-group) was separated from the other AA diploids. Finally (EGC/EC) seemed to be independent of agropedoclimatic conditions of banana growth.